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Congenital Platelet Dysfunction and von Willebrand Disease
Published in Harold R. Schumacher, William A. Rock, Sanford A. Stass, Handbook of Hematologic Pathology, 2019
Platelet lumi-aggregation studies were performed. As may be seen in Fig. 1, addition of a variety of agonists to the platelet-rich plasma (PRP) of a normal individual results in increased light transmission, which is reflected by a downward deflection in the aggregation channel (upper tracing in each panel). Stimulation of platelet secretion results in the release of ATP from the platelet’s dense granules. This released ATP then reacts with firefly luciferin and luciferase present in the cuvet in a light-emitting reaction. This light is detected by a second photodetector operating at a wavelength different from that used in the aggregation channel. ATP release is accordingly represented by an upward deflection in the secretion channel (lower tracing in each panel). At the end of each run, a known amount of ATP has been added to the cuvet, providing an internal standard for the secretion channel that may be used in quantitative determinations of the amount of ATP released.
New methodology for microbiological quality assurance
Published in R. M. Baird, S. F. Bloomfield, Microbial quality assurance in cosmetics, toiletries and non-sterile Pharmaceuticals, 2017
ATP detection has been investigated for sterility testing of pharmaceuticals (Bussey and Tsuji 1986) and for bacteriological screening of toiletries and cosmetics (Borovian 1991, Nielsen and Van Dellen 1989) and as such may have application across all three industries. Some general reviews of the technique have been reported by Stanley (1993), Stanley et al. (1989) and Szalay et al. (1993). The method for ATP analysis involves the firefly bioluminescence reaction (Fig. 14.4). This reaction uses one molecule of ATP to oxidize the substrate (firefly luciferin) in the presence of an enzyme (firefly luciferase) to produce one photon of light. There is a range of ATP instrumentation available that will monitor the light output from the reaction. Jago et al. (1989) reviewed the performance of ten luminometers. There have been a number of advances in recent years particularly with respect to automation and computer control and also with respect to portable instruments.
Introduction to Enzymes
Published in John C. Matthews, Fundamentals of Receptor, Enzyme, and Transport Kinetics, 2017
When no useful color, fluorescence, or size changes, etc., are available it is often possible to couple the reaction to other enzymes that will produce such changes. In coupled reactions the enzyme or substrate of interest is limiting in the reaction mixture. All the coupling reagents and enzymes are at saturating levels. An example of a coupled reaction is the enzyme hexokinase, which converts glucose plus ATP to glucose-6-phosphate plus ADP. After some reaction time has elapsed, a firefly luciferin-luciferase mixture is added to the hexokinase reaction mixture. The ATP that remains in the mixture reacts with the luciferin-luciferase to produce a flash of light. The intensity of the light flash is proportional to the amount of ATP that was present.
miR-340 Exerts Suppressive Effect on Retinoblastoma Progression by Targeting KIF14
Published in Current Eye Research, 2021
Hong-Kun Xu, Xiao-Dong Wang, De-Gong Wang, Dong-Dong Wei, Ling Liang, Chang-Hui Liu
The prediction results of bioinformatics software were further confirmed using the dual-luciferase reporter assay (Promega Corporation, Madison, WI, USA). Firstly, wild type plasmid WT KIF14 and mutant plasmid MUT KIF14 were constructed. After that, KIF14-WT or KIF14-MUT and miR-340 mimic or miR-340 mimic NC were co-transfected into RB cells. The transfected cells were seeded on 96-well plate at a density of 1 × 104/well for 48 h. Then 100 μL pyrolysis solution was added into each well to collect the supernatant after pyrolysis. Some of the supernatant was taken out and 40 μL firefly luciferase was added. The bioluminescence intensity was measured 15 s after homogeneous mixing. The bioluminescence intensity of the remaining supernatant was measured after adding 40 μL Renilla luciferase substrate and mixing homogeneously. Strength ratio of firefly luciferin and Renilla bioluminescence intensity values was calculated to reflect the influence of miR-340 on KIF14 level.
Comparison of platelet function tests for the in vitro quality assessment of platelet concentrates produced under real-life conditions
Published in Platelets, 2019
Lars Asmis, Anja Moldenhauer, Walter Hitzler, Peter Hellstern
Collagen-induced platelet ATP release was determined in accordance with the manufacturer’s instructions on a Lumi-aggregometer 700–2 from Chrono-Log, Havertown PA, USA, distributed by Probe & go, Lemgo, Germany. This system measures the increase in impedance between electrodes due to platelet aggregates and ATP secreted concurrently from platelet dense granules using a sensitive luminescent firefly luciferin-luciferase assay. Aggregation is recorded for 6 min, and maximum aggregation is expressed in ohms. ATP secretion was measured in nmol using Chrono-Lume luciferin-luciferase reagent and ATP standard. 475 µL of recalcified and Xipla-anticoagulated PRP were diluted with 475 µL of physiologic saline preheated to 37°C. After incubation for 3 min at 37°C, 50 µL of Chrono-Lume were added. After further incubation for 2 min at 37°C, 10 µL of collagen from equine tendon were added to obtain a final concentration of 5 µg mL−1 of collagen. The samples were stirred at 1200 rpm. Chrono-Lume luciferin-luciferase reagent and ATP standard were from Probe & go, Lemgo, Germany.
Integrated bioinformatics analysis of miRNA expression in Ewing sarcoma and potential regulatory effects of miR-21 via targeting ALCAM/CD166
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2019
Yuzhe Liu, Gaoyang Chen, He Liu, Zhaoyan Li, Qiwei Yang, Xinming Gu, Zhenwu Du, Guizhen Zhang, Jincheng Wang
To determine whether ALCAM was regulated by miR-21 through targeting its 3′-UTR, the reporter gene recombinant plasmids were first constructed, the wild type or mutant type ALCAM 3′-URT were cloned into the Kpnl and HindIII sites of the pGL6-miR vector (Beyotime, China). About 2 × 104 293 T cells were seeded into each well of 24-well plates for 24 h before transfection. Firefly luciferase reporter gene assay kit (Beyotime) was adopted for the luciferase reporter analysis following the manufacturer’s protocols, pGL6-ALCAM-wt and pGL6-ALCAM-mut were co-transfected with miR-21mimics or miR-21 mimics-control into 293 T cells. The maximum luminescence wavelength of luciferin catalysed by firefly luciferin is 560 nm.